Chitosan is a high molecular weight linear polymer composed of 2-amino-2-deoxy-D-gluose obtained by deacety-lation of chitin-nature's second most abundant polymer. The chitosan solution properties and solubility as well as its chemical and biological characteristics make it an attractive biopolymer for a variety of applications. Chitosan whose solubility is known to be pH dependent and which is a non-toxic, biodegradable polymer having chelation abilities and even some therapeutic properties, has been used before in cosmetics for both hair and skin care, in agriculture as a fertilizer, in pharmaceutical compositions as wound healing and as a drug delivery agent, and in biotechnology as a matrix for entrapment of enzymes and cells (ref. 1).
Enzyme and whole cell entrapment has been well recognized as an important tool for carrying out numerous biotransformations and biosynthetic processes (for comprehensive review see ref. 2). The entrapment of the biocatalyst offers reuse of catalytic systems in various reactor configurations in either continuous or repeated batch operations, with significantly improved operational stability.
Whole cell entrapment may be achieved via cell adsorption or binding onto the surface of an appropriate particulate support or by physical entrapment in hydrophylic gel beads (for review see refs. 3, 4). The most popular approach to entrap cells is gel entrapment, in which cells are suspended first in aqueous solution of a monomer or a prepolymer. Then the solution is gellified by physical or chemical change (e.g. cooling, pH change, introduction of polymerization initiator or crosslinking agent), thus physically entrapping the cells.
Polysaccharides of biological origin (cog. alginate, carrageenan and agarose) are the most common matrices for cell entrapment. In recent years more attention has been focused on the potential of using chitosan as a matrix for entrapment of enzyme systems and cells due to several of its unique characteristics. The use of chitosan for this purpose is based on the fact that it is soluble in aqueous solutions of organic acids but readily precipitates upon elevation of the pH above 6 or by addition of anions such as pyrophosphate, alginate and lauryl sulfate (ref. 5). Using this dependency the following procedure was developed to prepare gel matrices for entrapment of cells: the cells to be entrapped were suspended in an aqueous organic acid solution of chitosan (pH&lt;6), and the suspension was added dropwise into a precipitating solution containing pyrophosphate or alginate (refs. 5 to 7). The use of a precipitation solution containing high-molecular-weight counterions such as alginate, results in capsules while the use of low-molecular-weight counterions such as pyrophosphates affords millimeter size beads. The so-obtained bead shaped chitosan matrices are advantageous over gel matrices obtained with other polysaccharides because of their stability in the presence of phosphate buffers which is required in many processes involving enzymes and cells. In addition, chitosan matrices are stable to Na.sup.+ and K.sup.+ ions and are easily prepared at room temperature.
However, the use of chitosan has also some intrinsic drawbacks. Thus, for entrapment of biologically and chemically active materials one has to suspend the active material in an acidic solution which may damage pH sensitive materials such as cells or enzymes. Also, in many cases chitosan induces flocculation of cells (ref. 5). Chitosan has, moreover, antifungal activity which prevents its use for the entrapment of yeast cells (ref. 8).
Moreover, known methods for the encapsulation of active material in chitosan beads are based on a reversible gelation mechanism which results in beads with a relatively low mechanical and chemical stability. Therefore, entrapment of biologically and chemically active materials in chitosan beads are still limited (ref. 1).
It is the object of the present invention to provide an improved process for the entrapment of active materials in chitosan beads that have improved mechanical and chemical stability. It is a further object of the invention to provide a method by which pH sensitive active materials and active materials that are sensitive to contact with chitosan are adequately protected.